Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by 10^–7–10^–5 M morphine and by 10^–6–10^–4 M naloxone, but was accelerated by 10^–4 M morphine. Electrical field stimulation augmented tritium outflow. The overflow evoked per ppulse decreased as the frequency of stimulation was increased from 0.3 to 3 Hz, but remained approximately constant when it was further increased to 10 Hz. At frequencies of 0.3, 1, and 3 Hz, but not at 10 Hz, morphine in concentrations of 10^–7–10^–5 M depressed the stimulation-induced overflow of tritium. 10^–4 M morphine did not influence the overflow induced by stimulation at 0.3 and 1 Hz and increased that evoked by stimulation at 10 Hz. Naloxone (10^–6–10^–4 M) did not change the response to stimulation. In the presence of 10^–4 M naloxone, 10^–6 M morphine did not diminish, and 10^–5 M morphine even enhanced the stimulation-induced overflow of tritium. The inhibitory effect of 10^–6 M morphine was not reduced, after tyrosine hydroxylase had been blocked by -methyltyrosine-methylester. It is concluded that morphine through an action on specific opiate receptors inhibits the release of transmitter from cerebrocortical noradrenergic neurones evoked by nerve impulses. By an action unrelated to opiate receptors, morphine at high concentrations increases the stimulation-induced overflow of noradrenaline, presumably by inhibiting its re-uptake into nerve endings.     

Autoreceptors and α2-adrenoceptors at the serotonergic axons of rabbit brain cortex

Summary Slices of the rabbit occipito-parietal cortex were preincubated with ³H-serotonin and then superfused and stimulated electrically (2 min at 3 Hz). In the absence of drugs, the stimulation-evoked overflow of tritium was approximately 3% of the tritium content of the tissue. Unlabelled serotonin and 5-carboxamido-tryptamine, when administered in the presence of 6-nitroquipazine, reduced the evoked overflow of tritium. Their effects were antagonized by metitepin (apparent pA₂ value 8.1) and (±)-cyanopindolol (apparent pA₂ value 6.4). Metitepin, but not cyanopindolol, increased evoked tritium overflow; the effect of metitepin was greater in the presence than in the absence of nitroquipazine. The evoked overflow of tritium was also depressed by clonidine, an effect antagonized by idazoxan (apparent pA₂ value 7.0) but not by prazosin. Phenylephrine caused a decrease only at high concentrations that simultaneously accelerated basal tritium efflux. Prazosin and idazoxan did not change evoked tritium overflow, and phentolamine increased it significantly only when administered in the presence of (+)-oxaprotiline. Rauwolscine produced an inhibition that was prevented by metitepin. It is concluded that the serotonergic axons of the rabbit occipitoparietal cortex possess presynaptic, release-inhibiting serotonin autoreceptors and ₂-adrenoceptors. The receptors appear to receive an input of endogenous serotonin and, to a lesser extent, noradrenaline, under the conditions of these in vitro experiments.     

Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices

Summary The effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized ³H-5-HT were examined in rat hypothalamic slices. ³H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-³H-tryptophan in the presence of the various durgs tested. ACh (10^–5 M) in the presence of eserine (2×10^–4 M), and carbachol (10^–5 M) stimulated the release of ³H-5-HT. In contrast, oxotremorine (10^–5 M) reduced the ³H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10^–6 M) and d-tubocurarine (10^–6 M). It was not reduced by the muscarinic antagonists, atropine (10^–6 M) and scopolamine (10^–6 M). In fact, each of two antagonists added alone to the incubating medium enhanced ³H-5-HT release. The scopolamine (10^–6 M) stimulating effect on ³H-5-HT release was suppressed by d-tubocurarine (10^–6 M). Finally, the inhibiting effect of oxotremorine on ³H-5-HT release was not prevented by d-tubocurarine (10^–6 M) but was in the presence of atropine (10^–6 M) or scopolamine (10^–6 M).In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of ³H-5-HT and ³H-5-HIAA from l-³H-tryptophan and on the accumulation of l-³H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous ³H-5-HT in hypothalamic synaptosomes (P₂ fraction).These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of ³H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of ³H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.     

Cyanopindolol is a highly potent and selective antagonist at the presynaptic serotonin autoreceptor in the rat brain cortex

Summary Rat brain cortex slices preincubated with ³H-serotonin were superfused with physiological salt solution containing the serotonin uptake blocker DU 24565 (6-nitroquipazine). The effects of (±)-cyanopindolol and its enantiomers, of ICI 118,551 (erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol) and of isoprenaline on the electrically (3 Hz) evoked ³H overflow were studied.(±)-Cyanopindolol increased the evoked ³H overflow; this effect was prevented by preexposure to the previously characterized serotonin receptor antagonist metitepin. The concentration-response curve of unlabelled serotonin for its inhibitory effect on the electrically evoked ³H overflow was shifted to the right by (±)-cyanopindolol (apparent pA₂ value: 8.29), whereas that of noradrenaline (determined in the absence of DU 24565) was not affected (apparent pA₂ value: <6.0). The concentration-response curve of serotonin was also shifted to the right by (–)-cyanopindolol (apparent pA₂ value: 8.30) and (+)-cyanopindolol (6.83) but not by ICI 118,551 (<5.5). Isoprenaline (up to 10 mol/l; examined in the absence of DU 24565) did not influence the electrically evoked ³H overflow.The present results show that the presynaptic serotonin autoreceptor is blocked by cyanopindolol in a stereoselective way. This drug is 20 times more potent than metitepin as an antagonist at the presynaptic serotonin autoreceptor, and, in contrast to the latter, it does not act as an antagonist at the presynaptic ₂-adrenoceptor on the serotoninergic neurone.This study was supported by a grant of the Deutsche Forschungsgemeinschaft     

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines

Summary In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5–20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the -adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca²⁺ or the application of tetrodotoxin (TTX, 2×10^–6 M). FS also resulted in a reversible increase in the fractional efflux of tritium (³H) from rat pancreatic tissues preincubated with either ³H-noradrenaline or ³H-adrenaline. The effects of FS (5–20 Hz) on ³H efflux were abolished by TTX (2×10^–6 M). TTX had no effect on the enhancement of ³H efflux caused by elevation of external potassium concentration (high K⁺, 75 mM). Removal of superfusate Ca²⁺ completely abolished both the FS- and high K⁺-induced increases in ³H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca²⁺-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.     

The effect of putative peptide neurotransmitters on cutaneous vascular permeability in the rat

Summary Meta-chlorophenylpiperazine inhibited serotonin and noradrenaline uptake by synaptosomes to the same extent with IC₅₀ of 1.3×10^–6 M and 5.8×10^–6 M respectively. Dopamine uptake was lesss affected by meta-chlorophenylpiperazine (IC₅₀ of 2.2×10^–5 M). Unlike d-amphetamine and d-fenfluramine, the drug did not significantly increase monoamine release in synaptosomal preparations. On the other hand, metachlorophenylpiperazine showed an IC₅₀ of 620 nM in displacing ³H-5HT binding to brain membranes. Meta-chlorophenylpiperazine produced a dose-dependent reduction of food intake and this effect was prevented by a pretreatment with methergoline, a serotonin antagonist. The effect of metachlorophenylpiperazine was not modified by an intraventricular injection of 6-hydroxydopamine, electrolytic lesions of nucleus medianus raphe or ventral noradrenergic bundle, nor by a pretreatment with penfluridol, propranolol or phentolamine. The data suggest that the decrease of food intake induced by metachlorophenylpiperazine depends on its ability to act as a serotonin agonist in the brain. The specificity of the effects on serotonin suggests that this compound could prove an important tool for studies aimed at elucidating the functional role of serotonin in the central nervous system.     

Noradrenaline release from slices of the thalamus of normal and morphine-dependent rats

Summary The effect of morphine on potassium-induced stimulation of (³H)-noradrenaline release from slices of the rat thalamus was investigated. The in vitro addition of morphine (10^–6 M) significantly depressed potassium-induced tritium overflow by 42% and this was prevented by the prior addition of naloxone (3×10^–6 M) to the medium. The stimulation-evoked overflow of tritium from slices of the thalamus of morphine-dependent rats was not significantly different from normal controls. Addition of naloxone (10^–5 M) 10 min before exposure of the tissues to 20 mM K⁺ significantly enhanced noradrenaline release from dependent slices. The results suggest that the basic release mechanism may have adapted to the continuous presynaptic inhibition of release by morphine.     

Effect of extracellular dopamine on the release of dopamine in the rabbit caudate nucleus: Evidence for a dopaminergic feedback inhibition

Summary Slices of the head of the rabbit caudate nucleus were preincubated with 10^–7 M ³H-dopamine and then superfused, and the effect of unlabeled dopamine on the outflow of tritium was investigated. In most experiments, nomifensine was added throughout superfusion in order to block uptake of the unlabeled amine. Nomifensine was a potent inhibitor of the uptake of ³H-dopamine into rabbit caudate synaptosomes, with an IC₅₀ of 5·10^–8 M at a ³H-dopamine concentration of 4·10^–8 M.In the absence of nomifensine, unlabeled dopamine (10^–7 M and higher concentrations) accelerated the basal outflow of tritium from preincubated slices. 10^–5 M nomifensine strongly counteracted the acceleration. In the presence of nomifensine, unlabeled dopamine (10^–7 to 10^–6 M) caused a concentrationdependent decrease of the overflow of tritium evoked by electrical stimulation at 0.1 Hz. Chlorpromazine and haloperidol (in the presence of nomifensine) increased the stimulation evoked overflow and antagonized the inhibitory effect of dopamine.It is concluded that extracellular dopamine shares with other dopaminergic agonists the ability to inhibit action potential-evoked release of intraneuronal dopamine. The inhibition is mediated by specific receptors. The results support the hypothesis that previously released dopamine, by an action on these receptors, can inhibit further release of dopamine.     

Facilitation of serotonin (5-HT) release in the rat brain cortex by cAMP and probable inhibition of adenylate cyclase in 5-HT nerve terminals by presynaptic α2-adrenoceptors

Summary Stimulation-evoked tritium overflow was examined in superfused rat brain cortex slices (stimulus: electrical impulses; 3 Hz) and synaptosomes (stimulus: potassium 12 mmol/l) preincubated with ³H-5-HT. 1. In slices and synaptosomes, the evoked ³H overflow was facilitated by forskolin and 8-Br-cAMP, but was not affected by AH 21-132 (an inhibitor of cAMP phosphodiesterase; cis-6-(p-acetamidophenyl)-1, 2, 3, 4, 4 a,10b-hexahydro-8, 9-dimethoxy-2-methylbenzo [c] [1,6]-naphthyridine). In the presence of AH 21-132, the facilitatory effect of forskolin on evoked overflow was increased. 2. In slices, AH 21-132 or combined administration of forskolin plus AH 21-132 did not change the percentage of basal or evoked ³H overflow represented by unmetabolized ³H-serotonin (about 30% and 60%, respectively). 3. In slices, cocaine or 6-nitroquipazine, an inhibitor of serotonin uptake, did not influence the increase in evoked overflow produced by forskolin plus AH 21-132. Forskolin plus AH 21-132 did not alter the inhibitory effect of serotonin (examined in the presence of 6-nitroquipazine) and the facilitatory effect of metitepin (a serotonin receptor antagonist) on evoked ³H overflow, but considerably decreased the inhibitory effect of clonidine or B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepine). The present results suggest that the serotoninergic nerve terminals in the rat brain cortex are endowed with an adenylate cyclase, which is negatively coupled to the presynaptic ₂-adrenoceptors, but is not linked to the presynaptic autoreceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft. Part of the present results was reported at the 9th International Congress of Pharmacology, London 1984 (Schlicker et al. 1984) Send offprint requests to M. Göthert     

Phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex

Summary Possible antagonist effects of phentolamine at presynaptic serotonin autoreceptors were studied in slices of the occipito-parietal cortices of the rabbit and the rat. The slices were preincubated with ³H-serotonin and then superfused and stimulated electrically with single pulses or pulse trains. Nitroquipazine 1 mol/l, a compound that inhibits the high affinity neuronal uptake of serotonin, was present in the superfusion medium in all one pulse-experiments as well as in experiments in which the effect of unlabelled serotonin was examined.In rabbit cortical slices, unlabelled serotonin reduced the single pulse-evoked overflow of tritium. Its concentrationresponse curve was not changed by the selective ₂-adrenoceptor antagonist idazoxan 1 mol/l but was shifted to the right by phentolamine 1 and 10 mol/l. Phentolamine 10 mol/l also shifted to the right the concentration-inhibition curve of the selective 5-HT₁-receptor agonist 5-carboxamidotryptamine. When the slices were stimulated by trains of 30 pulses at 3 Hz, phentolamine 1 and 10 mol/l but not 0.1 mol/l increased the evoked overflow of tritium, the maximal increase amounting to 178%; its effect was enhanced in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l (a drug combination that, when given alone, slightly increased the evoked overflow of tritium). The serotonin receptor antagonist metitepin at concentrations of 0.01–1 mol/l also increased the overflow of tritium elicited by 30 pulses/3 Hz, the maximal increase amounting to 280%; its effect was potentiated in the presence of nitroquipazine 1 mol/l plus idazoxan 10 mol/l but was abolished or almost abolished in the presence of nitroquipazine 1 mol/l plus phentolamine 10 mol/l (a drug combination that, given alone, greatly increased the evoked overflow of tritium). When slices were stimulated by trains of 360 pulses at 3 Hz, there was no apparent antagonism of phentolamine 10 mol/l against the inhibitory effect of unlabelled serotonin. In rat brain cortex slices, unlabelled serotonin reduced the overflow of tritium elicited by 4 pulses delivered at 100 Hz. Again, phentolamine 10 mol/l shifted the concentration-response curve to the right.It is concluded that phentolamine blocks presynaptic serotonin autoreceptors in rabbit and rat brain cortex with pA₂ values of 6.44 and 5.95, respectively. Previous failures to detect the antagonistic effect against exogenous agonists were probably due to stimulation conditions that led to marked endogenous autoinhibition of serotonin release. At least the major part of the increase by phentolamine of the release of serotonin is due to autoreceptor blockade rather than blockade of the presynaptic a2-adrenoceptors at the cortical serotoninergic axons.Send offprint requests to N. Limberger at the above address     

Phencyclidine and ketamine: Comparison with the effect of cocaine on the noradrenergic neurones of the rat brain cortex

Summary In slices of the rat occipital cortex, the influence of phencyclidine and ketamine on the accumulation of ³H-noradrenaline and the subsequent outflow of tritium was investigated, and was compared with the effect of cocaine.-All three drugs inhibited the accumulation of tritium during incubation of the slices with ³H-noradrenaline. Phencyclidine was slightly, whereas ketamine was much less effective than cocaine.-All three drugs accelerated the spontaneous outflow of tritium from slices preincubated with ³H-noradrenaline. The acceleration caused by low concentrations probably reflects an inhibition of the re-uptake of spontaneously released ³H-noradrenaline; in addition, high concentrations (10^–4M phencyclidine, 3×10^–4–10^–3M cocaine and 10^–3–3×10^–3M ketamine) appear to release tritiated compounds from the neurones. The distance between uptakeinhibiting and releasing concentrations was much greater for cocaine than for phencyclidine and ketamine.-All three drugs enhanced the overflow of tritium evoked by electrical field stimulation. The increase probably reflects an inhibition of the re-uptake of released ³H-noradrenaline; in addition, phencyclidine appears to enhance the release of noradrenaline per pulse.-The actions of phencyclidine and ketamine on central noradrenergic neurones may contribute to the characteristic psychotropic side-effects of these general anaesthetics.     

Involvement of alpha-receptors in clonidine-induced inhibition of transmitter release from central monoamine neurones

Slices of rat cerebral cortex preincubated with (?)-³H-noradrenaline or ³H-5-hydroxytryptamine were stimulated by an electrical field, and the stimulation-induced overflow of tritium was determined. (1) Clonidine diminished the stimulation-evoked tritium overflow from slices preincubated with ³H-noradrenaline. The degree of this inhibition was greater at a low than at a high frequency of stimulation. (2) A high concentration of clonidine (10^?5 M) did not antagonize the increase of the stimulation-induced overflow caused by 10^?6 M or 10^?5 M cocaine, but abolished the increase caused by 10^?7 M of phentolamine or phenoxybenzamine. In the presence of cocaine, the inhibitory effect of clonidine was reduced. (3) 10^?5 M clonidine diminished the stimulation-evoked overflow of tritium from slices preincubated with ³H-5-hydroxytryptamine. (4) It is concluded that clonidine decreases, and phentolamine and phenoxybenzamine increase, the stimulation-induced release of noradrenaline from cerebral neurones by an activation and a blockade of α-receptors, respectively. A variety of secretory cells (secreting catecholamines, acetylcholine, 5-hydroxytryptamine, insulin or renin) seem to be endowed with structures similar to α-adrenergic receptors, which can modulate the secretion process.     

Cardiac electrophysiology of four neuroleptics: Melperone,haloperidol, thioridazine and chlorpromazine

Summary Effects of dopamine receptor agonists and antagonists on the release of dopamine were studied in the caudate nucleus of the rabbit. The nucleus contained 6.7 g/g of dopamine, but negligible levels of noradrenaline and dopamine--hydroxylase. No formation of ³H-noradrenaline was detected in caudate slices preincubated with ³H-dopamine, and more than 95% of the tritium content of the tissue consisted of ³H-dopamine.When caudate slices were preincubated with ³H-dopamine and then superfused with amine-free medium, there was a basal outflow of tritium that was not or only slightly changed by tetrodotoxin (10^–7 and 10^–6 M), apomorphine (up to 10^–5 M), bromocriptine (up to 10^–6 M), chlorpromazine (up to 10^–6 M), haloperidol (up to 10^–7 M), or omission of calcium. Electrical stimulation (3 Hz, 24 mA, 2 ms pulse duration, 2-min periods) greatly increased the outflow of tritium. The stimulation-evoked overflow was abolished by tetrodotoxin (10^–7 and 10^–6 M) and in calcium-free medium. Apomorphine (10^–8–10^–5 M) and bromocriptine (10^–8–10^–6 M) reduced, whereas chlorpromazine (10^–7 and 10^–6 M) and haloperidol (10^–8 and 10^–7 M) enhanced the evoked overflow. The inhibitory effect of apomorphine and bromocriptine was antagonized by chlorpromazine and haloperidol, but not by phentolamine.Silicone tubings that had been in contact with ³H-haloperidol retained tritiated material that was slowly eluted during perfusion with water or physiological salt solution. The material was identified as ³H-haloperidol. When silicone tubings pretreated with unlabelled haloperidol were used in subsequent dopamine release experiments, the inhibitory effect of apomorphine was not reproduced.It is concluded that, in the caudate nucleus of the rabbit, apomorphine and bromocriptine depress, whereas chlorpromazine and haloperidol facilitate action potential-evoked release of dopamine. The effects are mediated by specific receptors which may be located on the dopaminergic nerve terminals. The receptors appear to be normally activated by released dopamine itself, which thus inhibits its own further release. Part of the discrepancies in the literature concerning dopaminergic modulation of dopamine release may be due to retention of neuroleptic drugs in superfusion assemblies, followed by slow elution and interference with subsequent experiments.     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

Involvement of presynaptic H3 receptors in the inhibitory effect of histamine on serotonin release in the rat brain cortex

Summary Rat brain cortex slices or synaptosomes preincubated with ³H-serotonin were superfused with physiological salt solution (which, in the case of slices, contained citalopram, an inhibitor of serotonin uptake), and the effects of histamine and related drugs on the evoked tritium overflow were studied.The electrically (3 Hz) evoked tritium overflow from slices was inhibited by histamine and the H₃ receptor agonists R-(–)--methylhistamine and N-methylhistamine (pIC_12.5 values: 6.41, 7.28 and 6.12, respectively), but not affected by the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit (each at 10 mol/l). The concentration-response curve for histamine was shifted to the right by the H₃ receptor antagonists impromidine, burimamide and thioperamide (apparent pA₂ values: 7.45, 5.97 and 7.88, respectively); the concentration-response curve of serotonin for its inhibitory effect on the electrically evoked overflow was not affected by the three drugs (apparent pA₂ values: < 5.5, < 5.5 and < 6.5). Given alone, impromidine, thioperamide and a low concentration of burimamide facilitated the electrically evoked overflow. In slices superfused with K⁺-rich, Ca²⁺-free solution containing tetrodotoxin throughout and in synaptosomes superfused with Ca²⁺-free solution, histamine inhibited the overflow evoked by introduction of Ca²⁺ (in synaptosomes, simultaneously with an increased amount of K⁺). In either tissue, the effect of histamine was counteracted by thioperamide.The results provide evidence that exogenous and probably also endogenous histamine inhibits serotonin release in the rat brain cortex via presynaptic histamine H₃ receptors.Send offprint requests to E. Schlicker at the above address     

Evidence for negative feedback control of the release of [3H]serotonin from superfused mouse cerebellum slices induced by electrical field stimulation

Slices of mouse cerebellum preloaded with [3H]serotonin were superfused with a solution of Krebs-Ringer phosphate. The effects of exogenous serotonin, serotonin antagonists, fluoxetine, Ca2+ absence, Ca2+ chelation and frequency of stimulation on basal and electrically induced tritium overflow were investigated. Exogenous unlabeled serotonin decreased the stimulus-evoked tritium overflow in a concentration-related manner. This effect was blocked by simultaneous administration of methiothepin, but not by methysergide. When given alone, methiothepin did not alter the electrically induced tritium overflow at 50 Hz, but did potentiate the increased tritium overflow produced at 100 Hz. The basal tritium efflux was increased by exogenous serotonin, but this effect was reversed by the simultaneous administration of fluoxetine. Under this condition exogenous serotonin reduced the basal tritium efflux in a concentration-related manner. Superfusion of the slices with a Ca2+-free solution alone or in the presence of EGTA, reduced the basal tritium efflux and the stimulated tritium overflow. These results support the existence of serotoninergic presynaptic inhibitory autoreceptors in the cerebellum of the mouse.     

Modulation by drugs of the release of total tritium and 3H-5-HT from rat hypothalamic slices

Summary The release of total tritium and ³H-5hydroxytryptamine (5-HT) evoked by electrical stimulation from prelabelled rat hypothalamic slices was studied. Lysergic acid diethylamide (LSD) decreased while methiothepin increased both total tritium and ³H-5-HT overflow. The proportion of total tritium present as ³H-5-HT was equivalent under control conditions and in the presence of methiothepin and slightly increased in the presence of LSD. Whereas the addition of the 5-HT uptake blocker, citalopram, did not modify the evoked release of total tritium, the monoamine oxidase inhibitor, pargyline, decreased it. In the presence of either of these drugs the proportion of ³H-5-HT was increased. In the presence of citalopram, the inhibition by LSD was reduced on both the release of total tritium and of ³H-5HT. It thus appears that changes in electrically evoked total tritium overflow in general reflect changes in ³H-5-HT release. When uptake inhibitors or monoamine oxidase inhibitors are present in the medium, the situation is, however, more complex and results from experiments measuring only the release of total tritium should be interpreted with caution. Send offprint requests to: C. Moret at the above address